Salmonella is a major public health problem. There are more than one billion new Salmonella infections of humans each year that lead to more than three million deaths. The problem is greatly exacerbated by the emergence of multi-drug resistant strains. In addition to public health concerns, S. Typhimurium is also studied because it is a model pathogen without parallel for dissecting basic pathogenic processes as it combines the advantages of excellent genetics with tractable animal models of infection. Salmonella is believed to exploit migratory host cells as Trojan horses to spread from the gastrointestinal (GI) tract to internal organs. Dendritic cells are one such cell type, which can reenter the bloodstream by traversing endothelium in the basal to apical direction in a normal host process referred to as reverse transmigration. Reverse transmigration is likely relevant to numerous infectious processes including the spread of pathogenic microbes from the GI tract, lung tissue and the oral mucosa to the systemic circulation. Not surprisingly, infected cells do not normally reverse transmigrate, presumably because this could create a serious bloodstream infection. The host likely has mechanisms to detect conserved microbial components and balances the need to resolve inflammation with inhibiting the spread of microbes appropriately. Salmonella Typhimurium exploits the reverse transmigration pathway to deeper tissue, manipulating the migratory properties of infected cells to enter the bloodstream within them directly from the GI tract as an unappreciated component of its pathogenesis. Salmonella achieves this in part by secreting the type III effector SrfH into infected cells to subvert the host protein TRIP6 to stimulate reverse transmigration. In Aim 1, we will employ a variety of biochemical and genetic tools to delineate mechanistically exactly how the SrfH/TRIP6 interaction promotes travel through the reverse transmigration pathway. As a mutant deficient in type III secretion generally has a larger defect in triggering the reverse transmigration of infected cells than a srfH mutant, additional microbial and host factors must be involved. In Aim 2, we will utilize an in vitro revere transmigration assay along with murine intra-host dissemination experiments to determine the impact of the 9 host factors that are known to regulate the reverse transmigration of uninfected cells on the reverse transmigration of ones infected with Salmonella. Also, we will determine if these molecules are perturbed during infections. Finally, we will utilize the in vitro assay to identify the additional type III effector(s) involved in exploiting the reverse transmigration pathway to the bloodstream.